Differentiation of human induced pluripotent stem cells to megakaryocyte lineage by using 3D bioreactor, microfluidic system and acellular rat lung

Abstract

• Acellular rat lung as a natural scaffold can improve cell differentiation. • Polydimethylsiloxane made bioreactor provides a natural microenvironment for cells. • Microfluidic culture system can reduce the cost of cell culture & differentiation. Induced pluripotent stem cells (hiPSCs) are reprogrammed cells that can develop into all human cell types, including megakaryocytes. Extracellular matrix plays a crucial role in the differentiation of hiPSCs into megakaryocytes. Therefore, we aimed to prepare a suitable natural acellular scaffold, and 3D bioreactor for in-vitro proliferation, and differentiation of hiPSCs into megakaryocytes. The rat lung was extracted, and the decellularization process was performed in order to eradicate cellular and nuclear materials, and lung three-dimensional (3D) structure with the protein contents remained intact. Scanning electron microscopy (SEM), hematoxylin and eosin (H&E), and 4′, 6-diamidino-2-phenylindole (DAPI) staining were used to verify tissue decellularization, and to ensure the integrity of the tissue structure. The 3D polydimethylsiloxane (PDMS) based bioreactor was designed, and the recellularization of the acellular lung was performed by hiPSCs. Decellularized rat lung vessels were used to deliver culture media as a microfluidic system. Differentiation of hiPSCs to megakaryocytes was assessed by RT-PCR and flow cytometry. H&E, DAPI staining, and SEM analysis confirmed the integrity of the 3D lung structure. Flow cytometry and RT-PCR analysis revealed the presence of megakaryocyte markers in differentiated cells. It seems that natural acellular scaffold and microfluidic 3D bioreactor provides a suitable natural cost-benefit microenvironment for hiPSCs differentiation into megakaryocytes.